Compositions and methods for in vitro assays of fibrin activity

ABSTRACT

The present invention provides compositions exhibiting in vivo activity of fibrin in an in vitro setting, in vitro assays comprising such compositions, methods of producing such compositions, and methods of using such compositions and assays. The compositions of the invention include molecules with the biochemical properties of 1) high affinity binding to fibrin receptors and 2) activation of cell-signaling systems comparable to that observed in vivo by fibrin. The fibrin compositions of the invention are compatible both in biochemical assays and cell-based assays, and thus useful for in vitro assays for screening of test agents that modulate cell activation and/or signaling pathways mediated by fibrin or associated with fibrin activity.

GOVERNMENT SUPPORT

This invention was made with government support under Grant NS052189awarded by the National Institutes of Health. The United StatesGovernment may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the field of compositions and in vitroassays for the identification of agents for the prevention and treatmentof pathological conditions associated with neuronal injury.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an admission of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Fibrinogen is a key player in the coagulation cascade and has criticalfunctions in the regulation of inflammation (Davalos and Akassoglou,Semin. Immunopathol. 2012 34(1):43-62). Although soluble fibrinogen inthe bloodstream is not proinflammatory, its conversion to insolublefibrin after activation of coagulation leads to exposure within thefibrinogen y chain of the cryptic epitope y377_395, which binds toCD11b/CD18 integrin (Mac-1, complement receptor 3, aM˜2) (Altieri etal., J Biol Chem. 1990 Jul. 25; 265(21):12119-22; Lishko et al., 2002;Biochemistry. 2002 Oct. 29; 41(43):12942-51.). Epitope exposure canoccur during fibrin formation in vivo, or upon fibrinogen immobilizationon substrates. However, there is no method described to efficientlyreproduce the in vivo configurations of fibrin provided by itsinteractions with other molecules in vitro, or to predict the functionalactivity of an agent that binds to fibrin based on an in vitro assay.

There is thus a need in the art for a method of preparing fibrincompositions with the ability to predict functional utility usingactivity demonstrated in an in vitro setting. The present inventionaddresses this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present invention provides compositions exhibiting in vivo activityof fibrin in an in vitro setting, in vitro assays comprising suchcompositions, methods of producing such compositions, and methods ofusing such compositions and assays. The compositions of the inventioninclude molecules with the biochemical properties of 1) high affinitybinding to fibrin receptors and 2) activation of cell-signaling systemscomparable to that observed in vivo by fibrin. The fibrin compositionsof the invention are compatible both in biochemical assays andcell-based assays, and thus useful for in vitro assays for screening oftest agents that modulate cell activation and/or signaling pathwaysmediated by fibrin or associated with fibrin activity.

The fibrin compositions and assays of the invention are useful inassessing the predicted in vivo activity of test agents identified asmodulating fibrin-induced cell signaling and/or cell activation activityin an in vitro setting.

In one aspect, the invention provides an in vitro fibrin compositionthat displays high affinity binding to fibrin receptors and at least atwo-fold increase in cell activation upon introduction of thecomposition to cells in vitro. Preferably, the composition displaysbetween a 2- to 100-fold increase in cell activation upon introductionof the composition to cells in vitro.

Cell activation may be identified using a variety of biological endpoints, including but not limited to changes in cell size, changes ingene expression, ROS release, and changes in intracellular signalingpathways, such as signaling through NF-κB. In certain specific aspects,the cell activation is activation of an immune cell, e.g., a macrophage.In other specific aspects, the cell activation is activation of a cellof the central nervous system, e.g. a microglial cell. In other specificaspects, the cell activation is activation of CNS glia, such asoligodendrocytes or astrocytes. In other aspects, the cell activation isactivation of endothelial cells, neurons, or neuronal precursors. Inother aspects, cell activation can include non-CNS cells, such ascardiac, skin, liver, or stem cells.

The cellular activation displayed upon introduction, of the fibrincomposition of the invention to a cell population of interest may beassociated with an increase in gene expression. Alternatively or inaddition, the cellular activation displayed upon introduction of thefibrin composition of the invention to a cell population of interest maybe associated with an increase in reactive oxidative species.Alternatively or in addition, the cellular activation may be associatedwith changes in cell size or outgrowth of cell protrusions.Alternatively or in addition, the cellular activation may be associatedwith changes in intracellular signaling pathways, such as ERK1/2 orNF-κB. Alternatively or in addition, the cellular activation may beassociated with changes in cell differentiation. Alternatively or inaddition, the cellular activation may be associated with changes inprotein expression either on the cell surface or secreted in thesupernatants.

In certain embodiments, the invention provides an assay apparatuscomprising a solid surface and a fibrin composition of the invention,where the fibrin composition is adhered to the support surface. Thesupport surface to which the fibrin composition is immobilized may beany solid substance to which fibrin will adhere. Preferably, the solidsubstrate is glass, polyethylene, polystyrene or a cyclic olefin polymersubstrate.

In specific embodiments, the invention provides a research tool for theidentification of agents that modulate in vivo fibrin activity,comprising a fibrin composition that displays high affinity binding tofibrin receptors and at least a two-fold, and preferably between a 2 to100 fold increase in cell activation upon introduction of thecomposition in vitro to cells of the neuronal lineage.

In other embodiments, the invention provides an assay method foridentification of test agents that induce cell activation, comprisingimmobilizing a fibrin composition of the invention to a solid supportsurface, introducing cells of a neuronal lineage to the solid supportsurface, and measuring one or more biological outputs associated withthe activation of the cell. In certain aspects the cells activated arecells of the central nervous system. In other aspects the cellsactivated are immune cells. The biological outputs that can be measuredin these assays include, but are not limited to, expression of one ormore genes known to be associated with activation of the cells, and theproduction of a reactive oxidative species associated with activation ofthe cells.

In still other embodiments, the invention provides assay methods foridentification of test agents that modulate CD11b1 signaling throughfibrin, comprising the steps of immobilizing a fibrin composition ofclaim 1 to a solid support surface, introducing the test agent to thesupport surface, measuring the binding affinity of the test agent to thefibrin composition immobilized on the solid support surface, andidentifying agents that exhibit a high binding affinity to the fibrincomposition in vitro.

In yet other embodiments, the invention provides assay methods foridentification of an agent for modulation of a biological processinvolving signaling through fibrin, said method comprising providing aresearch tool comprising a fibrin composition of the invention adheredto a solid surface, testing one or more binding partners for modulationof functional activity of the fibrin in the research tool composition,and isolating the binding partners that display the desired change infunctional activity of the research tool composition. The bindingpartners that display the desired change in functional activity of theresearch tool composition are confirmed as candidates agents formodulation of a biological process associated with signaling throughfibrin.

Such candidate agents may be particularly useful in clinicalapplications for conditions involving impaired immune function, e.g.,diseases associated with autoimmunity such as multiple sclerosis orrheumatoid arthritis, or conditions associated with neuronal injury,e.g., neurodegeneration associated with aging, neurodegenerativediseases, and trauma (e.g., ischemia).

In one aspect, the invention provides methods to prepare in vitro fibrinthat mimics the biochemical and proinflammatory properties of the fibrinmatrices found in vivo.

In another aspect, the invention provides an apparatus comprising thefibrin composition of the invention for use in assay systems.

In yet another aspect, the invention provides methods of producing thefibrin compositions, apparatus comprising such fibrin compositions andresearch tools comprising such fibrin compositions.

In yet another aspect, the invention provides assay systems that use thefibrin compositions of the invention.

These and other aspects, features and advantages will be provided inmore detail as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the expression of Clcx10 mRNA in a firstassay system of the invention comprising the use of bone marrow derivedmacrophages (BMDMs).

FIG. 2 is a graph illustrating the expression of Ccl5 mRNA in a firstassay system of the invention comprising the use of BMDMs.

FIG. 3 is a graph illustrating the expression of I1-10 mRNA in a firstassay system of the invention comprising the use of BMDMs.

FIG. 4 is a graph illustrating the expression of Mcp-1 mRNA in a firstassay system of the invention comprising the use of BMDMs.

FIG. 5 is a graph illustrating the levels of reactive oxygen speciesresulting from dihydroethidium (DHE) oxidation in macrophages after 24hours exposure to the fibrin composition of the invention, alone or withIgG2b or anti-Mac 1 antibodies.

FIG. 6 is a graph illustrating the levels of reactive oxygen speciesresulting from DHE oxidation in macrophages after 48 hours exposure tothe fibrin composition of the invention, alone or with IgG2b or anti-Mac1 antibodies.

FIG. 7 is a graph illustrating binding of various assay substrates tothe proinflammatory fibrin receptor CD11b-1 domain using an ELISA assay.

FIG. 8 is a graph illustrating binding of fibrin and fibrinogen to theproinflammatory fibrin receptor CD11b-1 domain using an ELISA assay.

DETAILED DESCRIPTION OF THE INVENTION

The methods described herein may employ, unless otherwise indicated,conventional techniques and descriptions of molecular biology (includingrecombinant techniques), cell biology, biochemistry, and microarray andsequencing technology, which are within the skill of those who practicein the art. Such conventional techniques include polymer arraysynthesis, hybridization and ligation of oligonucleotides, sequencing ofoligonucleotides, and detection of hybridization using a label. Specificillustrations of suitable techniques can be had by reference to theexamples herein. However, equivalent conventional procedures can, ofcourse, also be used. Such conventional techniques and descriptions canbe found in standard laboratory manuals such as Harlow and Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, 1988; Sambrook and Russell, Molecular Cloning: A Laboratory Manual(2002) (all from Cold Spring Harbor Laboratory Press); Stryer, L.,Biochemistry (4th Ed.) W.H. Freeman, New York (1995); Lehninger,Principles of Biochemistry, 3^(rd) Ed., W. H. Freeman Pub., New York(2000); and Berg et al., Biochemistry, 5^(th) Ed., W.H. Freeman Pub.,New York (2002), all of which are herein incorporated by reference intheir entirety for all purposes. Before the present compositions,research tools and methods are described, it is to be understood thatthis invention is not limited to the specific methods, compositions,targets and uses described, as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to limit thescope of the present invention, which will be limited only by appendedclaims.

It should be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “atest agent” refers to one, more than one, or mixtures of such agents,and reference to “a method” includes reference to equivalent steps andmethods known to those skilled in the art, and so forth.

Where a range of values is provided, it is to be understood that eachintervening value between the upper and lower limit of that range—andany other stated or intervening value in that stated range—isencompassed within the invention. Where the stated range includes upperand lower limits, ranges excluding either of those included limits arealso included in the invention.

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing the formulations and methodologiesthat are described in the publication and which might be used inconnection with the presently described invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

DEFINITIONS

The term “antibody” is intended to include any polypeptidechain-containing molecular structure with a specific shape that fits toand recognizes an epitope, where one or more non-covalent bindinginteractions stabilize the complex between the molecular structure andthe epitope. As antibodies can be modified in a number of ways, the term“antibody” should be construed as covering any specific binding memberor substance having a binding domain with the required specificity.Thus, this term covers antibody fragments, derivatives, functionalequivalents and homologues of antibodies, including any polypeptidecomprising an immunoglobulin binding domain, whether natural or whollyor partially synthetic. Where bispecific antibodies are to be used,these may be conventional bispecific antibodies, which can bemanufactured in a variety of ways (Bolliger and Winter, Curr OpinBiotechnol. 1993 4(4):446-9), e.g., prepared chemically or from hybridhybridomas, or may be any of the bispecific antibody fragments mentionedabove. It may be preferable to use scFv dimers or diabodies rather thanwhole antibodies. Diabodies and scFv dimers can be constructed withoutan Fc region, using only variable domains, potentially reducing theeffects of anti-idiotypic reaction. Other forms of bispecific antibodiesinclude the single chain “Janusins” described in Traunecker et al, EmboJ. (1991) 10(12):3655-9. Such antibodies also include CRAbs, which arechelating antibodies which provide high affinity binding to an antigen,D. Neri, et al. J. Mol. Biol, 246, 367-373, and dual-variable domainantibodies as described in Wu C et al., Nat Biotechnol. 2007 November;25(11):1290-7. Epub 2007 Oct. 14.

The term “diagnostic tool” as used herein refers to any composition orassay of the invention used in order to carry out a diagnostic test orassay on a patient sample. As a diagnostic tool, the composition of theinvention may be considered an analyte specific reagent, and as such mayform part of a diagnostic test regulated by a federal or state agency.The use of the compositions of the invention as a diagnostic tool is notintended to be related to any use of the composition in the developmentof therapeutic agents.

The term “microglial activation” as used herein can refer to processesassociated with innate activation or adaptive activation of themicroglia. Such activation may include morphological changes of themicroglial cells, including shortening of cellular processes andenlargement of their soma, as well as the release of proinflammatorycytokines and chemokines, reactive oxygen and/or nitrogen intermediates,proteinases and complement proteins, and upregulation of cell surfaceactivation antigens.

The term “neurodegeneration” refers to a physiological state caused byneuronal injury associated with neuronal loss and/or damage. In specificaspects, neurodegeneration refers to neuronal injury resulting inimpaired cognitive function.

The term “neuronal injury” as used herein refers to any damage ordysfunction exhibited by neurons, including but not limited to loss ofmyelin, dendrite retraction, dendritic spine density reduction, axonaldamage and neuronal death. A “neuronal condition” as used herein refersto any damage or dysfunction exhibited by neurons, including but notlimited to dendrite retraction, dendritic spine density reduction,axonal damage and neuronal death. The term “pharmaceutically acceptablecarrier” as used herein is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Suitable carriers are described in themost recent edition of Remington's Pharmaceutical Sciences, a standardreference text in the field, which is incorporated herein by reference.Preferred examples of such carriers or diluents include, but are notlimited to, water, saline, Ringer's solutions, dextrose solution, and 5%human serum albumin. The use of such media and agents is well known inthe art. Except insofar as any conventional media or agent isincompatible with the agents provided herein, use thereof in thecomposition is contemplated.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified, labeled or derivatized aminoacids, and polypeptides having modified peptide backbones.

The term “peptidomimetic” as used herein refers to a protein-like chaindesigned to mimic a peptide. They typically arise from modification ofan existing peptide in order to alter the molecule's properties. Forexample, they may arise from modifications to change a molecule'sstability, biological activity, or bioavailability.

The term “pharmacophore” is used herein in an unconventional manner.Although the term conventionally means a geometric and/or chemicaldescription of a class or collection of compounds, as used here the termmeans a compound that has a specific biochemical activity or bindingproperty conferred by the 3-dimensional physical shape of the compoundand the electrochemical properties of the atoms making up the compound.Thus, as used here the term “pharmacophore” is a compound and not adescription of a collection of compounds which have definedcharacteristics. Specifically, a “pharmacophore” is a compound withthose characteristics.

The term “small molecule” refers to a molecule of a size comparable tothose organic molecules generally used in pharmaceuticals. The termexcludes biological macromolecules (e.g., proteins, nucleic acids,etc.). Preferred small organic molecules range in size up to about 5000Da, more preferably up to 2000 Da, and most preferably up to about 1000Da.

“Solid support” and “support” are used interchangeably and refer to amaterial or group of materials having a rigid or semi-rigid surface orsurfaces.

The term “research tool” as used herein refers to use of any animalmodel or assay of the invention for scientific enquiry, whether academicor commercial in nature, including the identification and development ofcandidate therapeutic agents. The research tools of the invention arenot themselves intended to be therapeutic or to be subject to regulatoryapproval; rather, the research tools of the invention are intended tofacilitate research and aid in such development activities, includingany activities performed with the intention to produce information tosupport a regulatory submission.

A “test agent” as used herein refers to any agent that is a candidate totreat a disease or symptom thereof. Such agents include, but are notlimited to, peptides; proteins (including derivatized or labeledproteins); antibodies or fragments thereof; small molecules; aptamers;carbohydrates and/or other non-protein binding moieties; derivatives andfragments of naturally-occurring binding partners; peptidomimetics; andpharmacophores.

As used herein, the terms “treat,” “treatment,” “treating,” and thelike, refer to obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment,” as used herein,covers any treatment of a disease in an animal, particularly in a human,and includes: (a) preventing the disease from occurring in a subjectwhich may be predisposed to the disease but has not yet been diagnosedas having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; and (c) relieving the disease, e.g., causing regression ofthe disease, e.g., to completely or partially remove symptoms of thedisease.

The Invention in General

The present invention is based on the novel development of a compositionthat mimics the biochemical and proinflammatory properties of fibrinmatrices found in vivo during pathological conditions in an in vitrosetting. These fibrin compositions are highly reproducible in activity,and provide the advantage of essentially mimicking the in vivo activityof fibrin, and use of such compositions in assays enable the screeningof fibrin inhibitors that have a higher likelihood of being efficaciousin clinical use. More particularly, by using a fibrin composition withsimilar properties of fibrin in vivo activity observed in pathologicalconditions, the probability of finding biologically active inhibitorswith in vivo efficacy is enhanced.

Assays using the fibrin compositions of the invention are useful in highthroughput screening applications, including assays to screen smallmolecules and antibody libraries for activity that modulates fibrinactivity in vivo. In addition, the assays of the invention can be usedto identify biomarkers indicative of fibrin-mediated pathologicalconditions, e.g., biomarkers present in the serum or cerebral spinalfluid (CSF) of a patient tested for a fibrin-mediated pathologicalcondition. In certain embodiments, the assays of the invention caninclude other biological molecules that may enhance or reduce fibrinactivity. Such biological molecules can include, but are not limited to,isolated fibrinogen, including fibrinogen that has undergonepost-translational modifications, such as citrullination ornitrosylation.

The invention also provides methods to prepare fibrin compositions thatmimic the biochemical and proinflammatory properties of the fibrinmatrices found in vivo during pathological conditions. The method offibrin preparation is highly reproducible. The assays described here arealso superior to any other in vitro assay preparations available interms of reproducibility and biochemical properties of high affinitybinding to fibrin receptors and/or signaling through fibrin, as well asat least a two-fold, and preferably between a 2 to 100 fold increase ofcell activation.

The fibrin compositions of the invention provide assays with the abilityto induce a significant increase in assay end points such as measuredincreases in cell signaling activity and/or gene expression. Forexample, the fibrin compositions of the invention can increase geneexpression in neuronal cells such as macrophages. The fibrincompositions of the present invention can also induce ROS release inmacrophages in a CD11b-dependent manner. These results indicate thatfibrin is responsible for the in vivo activity of fibroinogen-mediatedcell activation, and thus immobilized fibrin is the primary in vitrobioactive and proinflammatory substrate in the cell activation assays ofthe invention.

Fibrin and Fibrinogen Signaling Systems

Fibrin and fibrinogen have been shown to have a vast signaling capacitywith involvement in inflammatory mechanisms of a wide range of disordersincluding rheumatoid arthritis, sepsis, myocardial infarction, infectionand multiple sclerosis (MS) (Adams, R. A. et al., 2007, CurrentMedicinal Chemistry, 14(27), p. 2925-36). These broad receptor signalingactivities leading to various physiological reactions and differentialinduction of cytokines and chemokines suggest that fibrinogen and itsproteolytic product fibrin may be able to promote diverse responsesduring different stages of inflammation as well as having a role in theinhibition of adult neurogenesis. Fibrinogen and/or fibrin have beenimplicated in the interaction with an array of receptors on differingcell types, and thus the fibrin compositions, assay methods, apparatusand research tools of the invention are useful in identifying testagents that have activity in the various signaling pathways that areassociated with fibrin activity.

The CD11b/CD18 (αMβ2, or MAC-1) receptor is a member of the β-integrinfamily of receptors and controls the innate immune response byregulating phagocyte adhesion, migration and engulfment of potentiallyharmful factors (Ehlers, M R W., 2000 Microbes and Infection, 2(3), pp.289-294). The receptor is expressed on microglia cells, and upregulationhas been observed in the brains of post mortem Alzheimer's diseasepatients (Akiyama, H. & McGeer, P L, 1990. Journal of Neuroimmunology,30(1), pp. 81-93.). The association of fibrinogen with CD11b/CD18 onmany immune cells suggests an important role for fibrin-mediatedreceptor activity in inflammatory responses. Fibrinogen-mediated TNFαrelease in isolated peripheral blood monocytes was dependent on CD11b(Fan, S T and Edgington, T S, 1993, Journal of Immunology 150(7), pp.2972-80). Furthermore, fibrinogen treatment of the human monocytic cellline U937 induced significant IL-1β expression via CD11b and NFκBsignalling (Perez et al. 1999, supra). Finally, blocking fibrinformation with anticoagulant treatment or genetically eliminating thefibrinogen binding motif recognized by the microglial integrin receptorCD11b/CD18 inhibits perivascular microglial clustering and axonaldamage. Davalos D et al., Nat Commun. 2012; 3:1227.

Another integrin receptor CD11c/CD18, has been shown to interact withfibrinogen in the activation of dendritic cells (Ugarova, T P &Yakubenko, V P, 2001. Annals of the New York Academy of Sciences, 936,pp. 368-85). Studies using primary peritoneal macrophages have alsoshown fibrinogen can induce chemokine secretion via toll-like receptor 4(TLR4) (Smiley, S T. et al., Journal of Immunology, 167(5), pp.2887-2894). High plasma levels of fibrinogen have been linked to theonset of dementia and increase the risk of AD (Xu G et. al.. Int J ClinPract 62(7): 1070-1075; van Oijen M et. al., Stroke 36(12): 2637-2641).Fibrinogen has also been associated with the av33 integrin receptor,causing inhibition of neurite outgrowth via phosphorylation of theepidermal growth factor (EGF) receptor (Schachtrup C et al., 2010.Journal of Neuroscience, 30(17), pp. 5843-5854.). Fibrinogen has beenshown to serve as an early signal in glial scar formation via theTGFβ/Smad signaling pathway in astrocytes. Schachtrup, C. et al., 2010.Id.

Test Agents for Screening Using the Compositions and Apparatus of theInvention

The fibrin compositions of the invention are useful as either researchor diagnostic tools in functional assays, including: assays used tounderstand physiological processes; assays to identify new bindingpartners (including drug candidates) that selectively bind to fibrinsignaling complexes and modulate specific signaling processes; andassays to test known compounds (including synthetic, recombinant ornaturally-occurring compounds) for their effect on signaling through thefibrinogen/fibrin pathways, and the like. It is known in thepharmaceutical arts that binding affinity to a target and efficacy donot necessarily correlate, and that identification of functional changesconferred by a binding partner is a much better predictor of efficacythan binding affinity alone. The fibrin compositions of the inventionare especially powerful in identification of binding partners withfunctional activity rather than just affinity, as the compositions notonly can recreate functional activity of fibrin but also providepotentiation of the signaling pathway through interaction of the fibrinand at least one binding partner.

The assays for use with the compositions of the present inventioninclude biochemical assays which can be correlated with in vivo efficacyfor a physiological process, ex vivo cell-based assays for measurementof a physiological process, in vivo assays for direct or indirectmeasurement of a physiological process, etc.

The assays of the invention correlate with in vivo modulation ofsignaling through activated fibrin. Examples of cell-based assays foruse with the present invention include, but are not limited to, highthroughput binding screening; assays to measure cell activation,proliferation, necrosis and/or apoptosis; flow cytometry assays;metabolic assays measuring labeling or turnover; phase and fluorescencemicroscopy; receptor phosphorylation and/or turnover; cell signalingassays; immunohistochemistry studies; reporter gene assays, andsubcellular fractionation and localization. More specific examples ofsuch assays are: FLIPR to detect changes in intracellular calciumconcentration and cell-based ELISA assays to detect and quantifycellular proteins including post-translational modifications associatedwith cell activation.

Biochemical assays can also be used to correlate binding with efficacyin the methods of the invention. These include, but are not limited to,spectrophotometric assays, fluorometric assays, calorimetric assays,chemiluminescent assays, radiometric assays, chromatographic assays,colorimetric assays, and substrate specificity inhibitor kinase assays.Specific examples are: luciferase assays, in which firefly luciferaseprotein catalyzes luciferin oxidation and light is generated in thereaction, and which is frequently used as a reporter gene for measuringpromoter activity or transfection efficiency; electrophoresis;gas-liquid chromatography; and Forster resonance energy transfer (FRET).

In a specific aspect, in vitro assays are utilized to provide acorrelation of binding affinity with efficacy in modulating a target.Examples of in vivo functional assays are radiolabelling assays,fluorescent protein expression assays, in vivo capture assays, NMRspectroscopy, or assays specifically designed to identify efficacy in ananimal model of a pathological process.

For example, in treatment of certain diseases or disorders, therapeuticsneed to be initially tested in in vivo models due to the complexphysiological parameters involved with efficacy.

The fibrin composition of the invention may also be used as a diagnostictool to detect the presence or absence of molecules associated with afibrin-mediated biological process in a patient.

Thus, in one aspect, the invention includes research tools comprisingthe compositions of the invention, and uses of such research tools inidentification, investigation and/or confirmation of activity of testagents that may be useful as therapeutic agents. These research toolsare generally useful before the inception of a plan for the clinicaldevelopment of a test agent in the treatment of a fibrin-associatedcondition. The research tools of the invention provide for theidentification and/or confirmation of the functional activity of anagent with fibrin binding sites.

Use of the research tools of the invention can in essence recreate oneor more functional fibrin interactions that occur in vivo in abiological process (e.g., binding of fibrin to CD11b1/CD18), thuspotentiating selective binding of test agents that will have in vivoactivity. It is known in the pharmaceutical arts that binding affinityto a target and in vivo modulation of a target do not necessarilycorrelate, and that identification of functional changes conferred by abinding partner is a much better predictor of efficacy than bindingaffinity alone. The fibrin compositions of the invention are especiallypowerful in identification of test agents with functional activityrather than just affinity to fibrin or fibrinogen.

The compositions and apparatus of the present invention aresignificantly advantageous over existing in vitro assays for functionalfibrin activity, as they provide the ability to screen for inhibitorsusing a proinflammatory substrate with similar properties as the onefound in disease, therefore increasing the probability of identifyinginhibitors with in vivo efficacy.

Moreover, the assays are reproducible and thus appropriate for highthroughput screening. For example, the assays can also be used to screensmall molecule and antibody libraries, and used for biomarker screeningfor autoantibodies in serum or CSF of patients. The plates can be storedin −20° C. until use allowing for scale up of screening plates andquality controls for HTS.

The assay can be modified to include preparations of purifiedfibrinogen, fibrinogen depleted from growth factors or IgG, orfibrinogen that has undergone post-translational modifications, such ascitrullination or nitrosylation. The assay can also be used to screenantibody libraries for identifying fibrin-specific antibodies.

To confirm the functional activity of a test agent, a therapeuticallyeffective amount of a test agent of the invention may be administered toa subject (including an animal model of a neurological pathology) toconfirm its in vivo activity following identification in an assay of theinvention. By “therapeutically effective dose or amount” or “effectiveamount” is meant an amount of the test agent that, when administered,brings about a positive therapeutic response with respect to neuronalinjury. In some embodiments of the invention, the therapeuticallyeffective dose is in the range from about 0.1 g/kg to about 100 mg/kgbody weight, about 0.001 mg/kg to about 50 mg/kg, about 0.01 mg/kg toabout 30 mg/kg, about 0.1 mg/kg to about 25 mg/kg, about 1 mg/kg toabout 20 mg/kg, about 3 mg/kg to about 15 mg/kg, about 5 mg/kg to about12 mg/kg, about 7 mg/kg to about 10 mg/kg or any range of value therein.It is recognized that the method of treatment may comprise a singleadministration of a therapeutically effective dose or multipleadministrations of a therapeutically effective dose.

The test agent is administered to supply a desired therapeutic dose topromote a desired therapeutic response of the modulator to thetherapeutic area. By “desired therapeutic response” is intended animprovement in the condition or in the symptoms associated with thecondition, including the inhibition of angiogenesis.

The test agents can be formulated in a unit dosage such as a solution,suspension or emulsion, in association with a pharmaceuticallyacceptable carrier. Such carriers are inherently nontoxic andnontherapeutic. Examples of such carriers are saline, Ringer's solution,dextrose solution, and Hanks' solution. Nonaqueous carriers such asfixed oils and ethyl oleate may also be used. The vehicle may containminor amounts of additives such as substances that enhance chemicalstability, including buffers and preservatives.

Various methods of delivery can be used to deliver the test agent, andwill in part be dependent upon the agent and its bioavailability. Forexample, small molecules or other agents that are bioavailable may beadministered orally, whereas protein-based agents are generally but notexclusively administered parenterally. Certain agents may beadministered systemically, while others may be more beneficial with alocal delivery. The method of delivery will be apparent to one skilledin the art upon reading the specification, and can be determined in viewof the specific properties of the test agent.

It is understood that the effective amount of a test agent may varydepending on the nature of the effect desired, frequency of treatment,any concurrent treatment, the health, weight of the recipient, and thelike. See, e.g., Berkow et al., eds., Merck Manual, 16th edition, Merckand Co., Rahway, N.J. (1992); Goodman et al., eds., Goodman and Oilman'sThe Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press,Inc., Elmsford, N.Y. (1990); Avery's Drug Treatment. Principles andPractice of Clinical Pharmacology and Therapeutics, 3rd edition, ADISPress, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi,Pharmacology, Little, Brown and Co., Boston (1985), Katzung, Basic andClinical Pharmacology, Appleton and Lange, Norwalk, Conn. (1992), whichreferences and references cited therein, are entirely incorporatedherein by reference.

The test agent may be contained in a pharmaceutically-acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. The use of such media and agents for delivering cells is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the cells or polypeptides provided herein, use thereofin the compositions is contemplated. Supplementary active compounds canalso be incorporated into the test agents.

For confirming in vivo activity, a composition comprising a test agentis formulated to be compatible with its intended route ofadministration. Examples of routes of administration includeintravenous, intraarterial, intracoronary, parenteral, subcutaneous,subdermal, subcutaneous, intraperitoneal, intraventricular infusion,infusion catheter, balloon catheter, bolus injection, direct applicationto tissue surfaces during surgery, or other convenient routes.

Solutions or suspensions used for such administration can include othercomponents such as sterile diluents like water for dilution, salinesolutions, polyethylene glycols, glycerin, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Thecomposition can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Test agents suitable for injectable use include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, or phosphate buffered saline(PBS). In all cases, the composition must be sterile and should be fluidto the extent possible. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents.

Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin. Sterile injectable solutionscan be prepared by incorporating an active agent in the required amountin an appropriate solvent with a selected combination of ingredients,followed by filter sterilization. Generally, dispersions are prepared byincorporating an active agent into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. In many cases, it will be preferable to includeisotonic agents.

Fibrin Assay Apparatus

The fibrin compositions of the invention can be used to create variousconfigurations of apparatus for use in fibrin-mediated assays. In theassay apparatus of the invention, the fibrin compositions areimmobilized onto various solid supports.

In many embodiments, at least one surface of the solid support will besubstantially flat, although in some embodiments it may be desirable tophysically separate regions on the support with, for example, wells.Multiple different configurations can be used for the adhesion of thefibrin compositions of the invention, and can be tailored based on thedesired assay methodology and/or instrumentation.

The solid upon which the fibrin composition is supported is not limitedto a particular substrate. Various solid supports, including glass-basedand plastic-based, allow the solid support to be adapted to suit anyparticular assay. Solids which may be of use in this invention thusinclude, but are not limited to, silica-based substrates, such as glass,fused silica and other silica-containing materials and plastic materialssuch as polyethylene, polystyrene, poly(vinyl chloride), polypropylene,nylons, polyesters, polycarbonates and poly (methyl methacrylate).Preferred plastics materials are polyethylene, polystyrene and cyclicolefin polymer substrates. Alternatively, other solid supports may beused such as gold, titanium dioxide, or silicon supports.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example 1 Production of an Apparatus Comprising the Fibrin Compositionof the Invention

Fibrinogen solution was prepared using plasminogen-depleted fibrinogenfrom human plasma (Calbiochem) in cell culture grade dissolved inendotoxin-free HyClone™ water (ThermoFisher, Waltham, Mass.) to a finalconcentration of 25 mg/ml. The fibrinogen solution was prepared at 37°C., swirling every 30 minutes. The fibrinogen solution was added to a 20μM HEPES-buffered saline solution to make a 20 μg/ml fibrinogensolution. The solution was stored in 1 ml aliquots at −80° C. until use.

A thrombin solution was prepared using the HEPES dissolved in 0.9%saline to a concentration of 1 M. Thrombin (1 KU) was dissolved in 1 mlHyClone™ water, and stored in 20 μl aliquots at 80° C. A CaCl₂ solutionwas created by dissolving CaCl₂ in HyClone water to a concentration of0.7M CaCl₂.

In a separate tube, CaCl₂ solution was added to a 20 μM HEPES-bufferedsaline solution to a 1.4 μM final concentration. The thrombin solutionwas added to the CaCl₂ solution to create a solution with 2 U/ml ofthrombin.

A fibrinogen assay plate was created by dispensing 50 μl of 20 μMHEPES-buffered saline solution in each well of a 96-well plate (NUNCMaxiSorp). 50 μl of 20 μg/ml fibrinogen solution was added into wellscontaining HEPES-buffered saline.

A fibrin assay plate was created by dispensing 50 μl of 20 μMHEPES-buffered saline solution containing CaCl₂ and thrombin in eachwell of a 96-well plate (NUNC MaxiSorp). 50 μl of the 20 μg/mlfibrinogen solution was added into wells containing HEPES-bufferedsaline with CaCl₂ and thrombin. The plate was sealed with adhesive sealagainst evaporation and incubated at 37° C. for 1 hr. After incubation,the seal was removed and the plates stored overnight in a drying chamberat 37° C. Following incubation, the plates were sealed with adhesiveseal and stored at −20° C. or 4° C.

Example 2 Use of Fibrin Composition in an In Vitro Gene Expression Assay

To compare pro-inflammatory effects of fibrinogen and fibrin onmacrophage activation, a chemokine gene expression assay was performedon cultured bone marrow derived macrophages (BMDMs) in fibrinogen- andfibrin-coated plates. Fibrin and fibrionogen assay plates were preparedas generally described in Example 1. The wells of each assay plate werecoated with 100 μg/ml fibrinogen or 100 μg/ml fibrin. An uncoated96-well plate was also used to serve as a negative control.

Approximately 100,000 BMDMs were plated per well on the fibrin assayplate, the fibrinogen assay plate, and the control plate. The plateswere allowed to incubate for 6 hours at room temperature. Followingincubation, real time PCR analysis expression using isolated mRNA fromthe BMDMs was performed. The results of the gene expression aresummarized in FIGS. 1-4. The expression of Clcx10, Ccl5, I1-10 and Mcp-1mRNA was significantly elevated in the fibrin assay plates compared tothe mRNA of both the negative control plates and the fibrininogen assayplates. This demonstrates that the fibrin composition in the fibrinassay plates was able to stimulate a gene expression response inmacrophages that is significantly higher than that seen by exposure ofthe macrophages to fibrinogen.

Example 3 Detection of Reactive Oxygen Species Using the FibrinComposition in an In Vitro Assay

One of the most invariant features of activated microglia andmacrophages is the production of reactive-oxygen species (ROS). Thus,the generation of ROS by these cells is a reliable indicator of theiractivation.

To demonstrate that the fibrin composition of the invention had theability to generate ROS in vitro, and thus activate microglia and/ormacrophages, the fibrin assay plates produced using the methods ofExample 1 were tested for their ability to induce ROS from BMDMs.

The chemical dihydroethidium (DHE) is oxidized specifically bysuperoxide anions, and the oxidized dye fluoresces red. By measuring theintensity of fluorescent signals in the wells produced by the oxidationof DHE, the amount of superoxide produced in the wells can be deduced.To investigate the ability of the fibrin compositions of the inventionto activate macrophage, and thus to cause the release of ROS, BMDMs wereexposed to fibrin assay plates comprising the fibrin compositions of theinvention.

In addition, preliminary evidence suggests that at least some fibrinsignaling activity may be through CD11b/CD18 receptor activation byfibrin-induced ROS. To investigate this, BMDMs were also incubated inthe fibrin assay plates in the presence of an anti-CD11b antibody withthe ability to block signaling through CD11b.

Fibrin assay plates were created by dispensing 50 μl of 20 μMHEPES-buffered saline solution containing CaCl₂ and thrombin in eachwell of a 96-well plate (NUNC MaxiSorp). 50 μl of the 20 μg/mlfibrinogen solution was added into wells containing HEPES-bufferedsaline with CaCl₂ and thrombin. The plate was sealed with adhesive sealagainst evaporation and incubated at 37° C. for 1 hr. After incubation,the seal was removed and the plates stored overnight in a drying chamberat 37° C. Following incubation, the plates were sealed with adhesiveseal and stored at −20° C. or 4° C.

BMDMs were preincubated with 10 μM DHE for 30 min and then plated in thefibrin assays plates, or a control uncoated 96-well plate. A portion ofthese BMDMs were also incubated with either 10 μg/ml anti-Mac1(CD11b/CD18) antibody or 10 μg/ml IgG2b. BMDMs were incubated withantibodies for 30 min at room temperature prior to fibrin exposure.After 30 min antibody incubation, cells were removed from the plate byPBS+0.04% EDTA.

Approximately 100,000 of each of the three groups of DHE-treatedBMDMs—the DHE-treated BMDMs, the DHE-treated BMDMs incubated with IgG2band the DHE-treated BMDMs incubated with anti-Mac1 (CD11b/CD18)—wereplated per well on the fibrin assay plates and the control plate. Theresults are shown in FIGS. 5 and 6.

Statistical analysis was provided by 1 way ANOVA, using 8 wells/group.Levels of ROS as detected with DHE fluorescence were significantlyincreased in fibrin-stimulated macrophages. The fibrin incubated BMDMsshowed a significantly increased level of ROS production as compared tothe BMDMs treated with IgG2b or with anti-Mac1 antibodies 24 hours afterplating (FIG. 5). After 48 hours, the fibrin incubated BMDMs continuedto demonstrate a significantly increased level of ROS production ascompared to the BMDMs treated with anti-Mac1 antibodies 48 hours afterplating (FIG. 6).

Example 4 Binding of CD11b Using In Vitro Fibrin Compositions in an InVitro Reactive Oxygen Species Assay

Fibrin and fibrinogen assay plates were created as described inExample 1. These plates were incubated in blocking buffer containing PBSwith 5% BSA with 0.05% Tween-20 for 1 hour and sealed with adhesiveseal. Following incubation, the plates were washed five times with PBScontaining 0.05% Tween-20.

Fibrin or fibrinogen were incubated with 50 μL per well of 10 μM CD11b1domain, singly biotinylated at the amino-terminus in diluent containingPBS with 0.5% BSA and 0.05% Tween-20, for 2 hours at 37° C. The plateswere washed five times with PBS containing 0.05% Tween-20 and incubatedwith 1 μg/mL HRP-coupled Streptavidin (BD Pharmingen, San Jose, Calif.)diluted 1:1000 in PBS with 0.5% BSA and 0.05% Tween-20 for 1 hour atroom temperature. The plates were again washed five times with PBScontaining 0.05% Tween-20, and incubated with undiluted TMB/E substrate(Chemicon-Millipore, Temecula, Calif.), typically for 4 to 5 minutes atroom temperature. The reactions were stopped by the addition of 100μl/well of 1N HCl. Fluorescence was detected at 450 nm.

This experiment demonstrates a side-by-side comparison of the fibrincompositions and fibrin assay apparatus of the invention compared to invitro fibrinogen compositions of the invention. Compared to fibrinogen,the fibrin composition substrate exhibits superior binding to theproinflammatory fibrin receptor CD11b1-domain (FIGS. 7 and 8). Theexposure of the pro-inflammatory fibrin epitope in vitro provides betterprediction of functional results in vivo in the cell-based and testagent binding assays using the compositions and apparatus of theinvention.

The experimental results of Example 4 indicate that fibrin assay platesprovide an improved substrate for exposure of the CD11b/CD18 epitope,and thus an enhanced functional screening technique for identifyingagents that modulate CD11b1 signaling through fibrin.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements and equivalents which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein.

1.-18. (canceled)
 19. An assay method for identification of test agentsthat induce cell activation, comprising: immobilizing a fibrincomposition to a solid support surface, wherein the fibrin compositiondisplays: high affinity binding to fibrin receptors or crypticfibrinogen receptors; and at least a two-fold increase in cellactivation upon introduction of the composition to cells in vitro;introducing cells to the solid support surface; and measuring one ormore biological outputs associated with the activation of the cell. 20.The assay method of claim 19, wherein the cells introduced to the solidsupport are cells of the mammalian central nervous system.
 21. The assaymethod of claim 20, wherein the cells are microglia.
 22. The assaymethod of claim 19, wherein the cells introduced to the solid supportare immune cells.
 23. The assay method of claim 22, wherein the cellsintroduced to the solid support are macrophage.
 24. The assay method ofclaim 19, wherein the biological outputs comprise expression of one ormore genes known to be associated with activation of the cells.
 25. Theassay method of claim 20, wherein the biological output comprises theproduction of a reactive oxidative species.
 26. A fibrin compositionthat displays: high affinity binding to fibrin receptors and at least atwo-fold increase in cell activation upon introduction of thecomposition to cells in vitro.
 27. The composition of claim 26, whereinthe cell activation is activation of an immune cell.
 28. The compositionof claim 27, wherein the cell activation is activation of a cell of theneuronal lineage.
 29. The composition of claim 26, wherein the cellularactivation is associated with an increase in gene expression.
 30. Thecomposition of claim 26, wherein the cellular activation is associatedwith an increase in reactive oxidative species.
 31. An assay apparatuscomprising a solid surface; and a fibrin composition that displays highaffinity binding to fibrin receptors or cryptic fibrinogen receptors andat least a two-fold increase in cell activation upon introduction of thecomposition in vitro; wherein the fibrin composition is adhered to thesupport surface.
 32. The assay apparatus of claim 31, wherein thesupport surface comprises glass.
 33. The assay apparatus of claim 31,wherein the support surface comprises polyethylene, polystyrene or acyclic olefin polymer substrate.
 34. A research tool for theidentification of agents that modulate in vivo fibrin activity,comprising a fibrin composition that displays high affinity binding tofibrin receptors and at least a 2- to 100-fold increase in cellactivation upon introduction of the composition in vitro to cells of theneuronal lineage.
 35. An assay method for identification of test agentsthat modulate CD11b signaling through fibrin, comprising the steps of:immobilizing a fibrin composition to a solid support surface, whereinthe fibrin composition displays high affinity binding to fibrinreceptors and at least a two-fold increase in cell activation uponintroduction of the composition in vitro; introducing the test agent tothe support surface; measuring the binding affinity of the test agent tothe fibrin composition immobilized on the solid support surface; andidentifying agents that exhibit a high binding affinity to the fibrincomposition in vitro.
 36. A method for identification of a drugcandidate for modulation of a biological process involving signalingthrough fibrin, said method comprising: providing a research toolcomposition comprising: a fibrin composition adhered to a solid surface;testing one or more binding partners for modulation of functionalactivity of the fibrin in the research tool composition; and isolatingthe binding partners that display the desired change in functionalactivity of the research tool composition; wherein the binding partnersthat display the desired change in functional activity of the researchtool composition are candidate agents for modulation of a biologicalprocess associated with signaling through fibrin.